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Review ArticleBiotechnological and Agronomic Potential of EndophyticPink-Pigmented Methylotrophic Methylobacterium spp.

Manuella Nóbrega Dourado, Aline Aparecida Camargo Neves,Daiene Souza Santos, and Welington Luiz Araújo

Department of Microbiology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil

Correspondence should be addressed to Welington Luiz Araujo; [email protected]

Received 1 November 2014; Revised 31 December 2014; Accepted 29 January 2015

Academic Editor: Frederick D. Quinn

Copyright © 2015 Manuella Nobrega Dourado et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

The genus Methylobacterium is composed of pink-pigmented facultative methylotrophic (PPFM) bacteria, which are able tosynthesize carotenoids and grow on reduced organic compounds containing one carbon (C

1), such as methanol and methylamine.

Due to their high phenotypic plasticity, these bacteria are able to colonize different habitats, such as soil, water, and sediment, anddifferent host plants as both endophytes and epiphytes. In plant colonization, the frequency and distribution may be influencedby plant genotype or by interactions with other associated microorganisms, which may result in increasing plant fitness. In thisreview, different aspects of interactions with the host plant are discussed, including their capacity to fix nitrogen, nodule the hostplant, produce cytokinins, auxin and enzymes involved in the induction of systemic resistance, such as pectinase and cellulase, andtherefore plant growth promotion. In addition, bacteria belonging to this group can be used to reduce environmental contaminationbecause they are able to degrade toxic compounds, tolerate high heavy metal concentrations, and increase plant tolerance to thesecompounds. Moreover, genome sequencing and omics approaches have revealed genes related to plant-bacteria interactions thatmay be important for developing strains able to promote plant growth and protection against phytopathogens.

1. Methylobacterium Genus

Members of the Methylobacterium genus are classified as𝛼-proteobacteria and include 51 reported species (http://www.bacterio.net/methylobacterium.html), which are repre-sented in Figure 1; most of them (35) were reported in the last10 years and are closely phylogenetically related (Figure 1).M.organophilum is the type strain [1], although M. extorquensstrain AM1 (Table 1), isolated as an airborne contaminantgrowing on methylamine [2], is the most studied strain andhas been used as a model organism for this genus, includingfor plant interactions and methylotrophic metabolism stud-ies.

This genus is composed of Gram-negative bacteria thatare generally with pink pigmentation due to carotenoidsynthesis [3], rod shaped, strictly aerobic and able to growusing compounds containing only one carbon (C

1), such

as methanol and methylamine [4]. Thus, these bacteriaare denoted “pink-pigmented facultative methylotrophs”

(PPFMs).Themain characteristic of this group is the ability tooxidizemethanol using themethanol dehydrogenase enzyme(MDH). The mxaF gene encodes the large subunit of thisenzyme, which is key in methylotrophic metabolism and isused to study that group of bacteria [5, 6].

Members of theMethylobacterium genus occupy differenthabitats due to their great phenotypic plasticity, includingsoil, water, leaf surfaces, nodules, grains, and air [7–9]. Theycan also be opportunistic pathogens in human beings [10].Growing in meristematic tissue, they can reach populationsof 104 to 106 colony-forming units (CFU) per gram of planttissue [11]. In addition, they can form biofilms [12, 13] and usemethylotrophic metabolism as an adaptive advantage duringplant host colonization [14].

The association betweenMethylobacterium spp. and hostplants varies from strong or symbiotic [15] to weak orepiphytic [16] and to intermediate or endophytic [17]. Duringinteractions with plants, M. nodulans and M. radiotoleranshave been reported to be involved in nitrogen fixation and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 909016, 19 pageshttp://dx.doi.org/10.1155/2015/909016

2 BioMed Research International

M. fujisawaense AJ250801M. oryzae AY683045

M. phyllosphaerae EF126746M. radiotolerans D32227

M. tardum AB252208M. longum FN868949.1

M. mesophilicum AB175636M. brachiatum AB175649

M. hispanicum AJ635304M. gregans AB252200

M. dankookense FJ155589M. trifolii NR 108524.1

M. cerastii FR733885.1M. jeotgali DQ471331

M. marchantiae FJ157976M. bullatum FJ268657

M. komagatae AB252201M. aerolatum EF174498

M. persicinum AB252202M. gnaphalii AB627071

M. brachythecii NR 114329.1

M. haplocladii NR 114325.1

M. thuringiense NR 108523.1

M. aquaticum AJ635303M. tarhaniae JQ864432

M. platani EF426729M. variabile AJ851087

M. nodulans AF220763M. isbiliense AJ888239

M. rhodinum AB175644M. podarium AF514774

M. salsuginis EF015478M. suomiense AB175645M. extorquens D32224

M. aminovorans AB175629M. rhodesianum AB175642

M. thiocyanatum U58018

M. populi AY251818M. zatmanii AB175647M. chloromethanicum GU130527.1

M. dichloromethanicum AB175631.1M. oxalidis AB607860M. soli EU860984

M. iners EF174497M. adhaesivum AM040156

M. gossipiicola EU912445

Rhizobium aggregatum X73041

0.01

M. organophilum AB175638

Figure 1: Phylogenetic analysis of the 16S rDNA genes ofMethylobacterium spp. strains (sequences available in Ribosomal Database Projectquery and the NCBI database) using the Kimura model. There were a total of 1288 nucleotide positions in the final dataset, and Rhizobiumaggregatum served as an outgroup.

BioMed Research International 3

Table1:Sequ

encedgeno

mes

(depositedin

theN

CBId

atabase)of

Methylobacterium

strainsa

ndtheirg

enom

icfeatures.

Organism

Strain

GenBa

nkassembly

IDIsolationsource

Keycharacteris

tic(s)

Genom

esize

(Mpb

)CG

content

(%)

Gene

Num

bero

fplasmid

Sequ

encing

institu

te

M.extorqu

ens

AM1

GCA

000022685.1

Air

Growth

onmethylamineo

rmethano

l6.88

68.7

5065

4University

ofWashing

tonGenom

eCenter

M.extorqu

ens

DM4

GCA

000083545.1

Soilcontam

inated

with

halogenated

hydrocarbo

ns

Chloromethane

degrader

6.12

68.1

5851

2Genoscope

M.extorqu

ens

PA1

GCA

000018845.1

Arabidopsis

thaliana

Arabidopsis

thaliana

epiphyte

5.47

68.2

4956

⋅⋅⋅

DOEJointG

enom

eInstitute

M.extorqu

ens

CM4

GCA

000021845.1

——

6.18

68.2

5463

2DOEJointG

enom

eInstitute

M.extorqu

ens

DSM

1306

0GCA

000243435.2

Meristem

sofP

inus

sylve

stris

Pinu

ssylv

estris

epiphyte

6.67

68.30

6894

⋅⋅⋅

DOEJointG

enom

eInstitute

M.m

esophilicum

SR1.6

/6GCA

000364

445.1

Citru

ssinensis

branches

Citrus

endo

phyte

6.2

69.47

6052

⋅⋅⋅

Rede

Paraense

degeno

micae

proteomica

M.nodulan

sORS

2060

GCA

000022085.1

from

root

nodu

les

from

thelegum

eCrotalaria

Non

pigm

ented,fix

nitro

gen,

Crotalaria

nodu

latin

g7.7

868.9

7765

7DOEJointG

enom

eInstitute

M.populi

BJ001

GCA

000019945.1

Popu

lussp.

Popu

lusd

eltoidesx

nigraD

N34

endo

phyte

5.80

695492

2DOEJointG

enom

eInstitute

M.radiotoleran

sJCM2831

GCA

000019725.1

—Ra

dioresistantstrain,

fixnitro

gen,

nodu

late

plants

6.08

71.5

5839

8DOEJointG

enom

eInstitute

Methylobacterium

sp.

4-46

GCA

000019365.1

—Lotononisb

ainesi

nodu

latin

g,ph

otosynthetic

7.66

71.4

7145

2DOEJointG

enom

eInstitute

Methylobacterium

sp.

GXF

4GCA

000272495.1

grapevinex

ylem

fluids

Endo

phyticfro

mxylem

fluids

6.12

69.6

5976

⋅⋅⋅

RochesterInstituteo

fTechno

logy

Methylobacterium

sp.

MB2

00GCA

000333655.1

biogas

reactor

—5.77

68.9

5038

⋅⋅⋅

College

ofLife

Sciencea

ndTechno

logy,G

uang

xiUniversity

Methylobacterium

sp.

77GCA

000372825.1

Lake

Washing

ton

—4.66

66.7

4108

⋅⋅⋅

DOEJointG

enom

eInstitute


Table1:Con

tinued.

Organism

Strain

GenBa

nkassembly

IDIsolationsource

Keycharacteris

tic(s)

Genom

esize

(Mpb

)CG

content

(%)

Gene

Num

bero

fplasmid

Sequ

encing

institu

te

Methylobacterium

sp.

WSM

2598

GCA

000379105.1

Lotononisb

ainesii

Root

nodu

latin

gbacteria,

bacteria-plant-soil

association

7.67

71.2

6631

⋅⋅⋅

DOEJointG

enom

eInstitute

Methylobacterium

sp.

285M

FTsu5.1

GCA

000383455.1

—Ba

cteria-plant

association

6.62

715970

⋅⋅⋅

DOEJointG

enom

eInstitute

Methylobacterium

sp.

10GCA

000519085.1

Lake

Washing

ton

—4.98

66.7

4285

⋅⋅⋅

DOEJointG

enom

eInstitute

Methylobacterium

sp.

B1GCA

000333255.1

rices

hoot

—5.91

69.6

—⋅⋅⋅

Kazusa

DNA

Research

Institute

Methylobacterium

sp.

B34

GCA

000333475.1

rices

hoot

—6.93

70.4

—⋅⋅⋅

Kazusa

DNA

Research

Institute

Methylobacterium

sp.

88A

GCA

000376345.1

Lake

Washing

ton

—4.89

67.1

4274

⋅⋅⋅

DOEJointG

enom

eInstitute

Methylobacterium

sp.

L2-4

GCA

000454305.1

Jatro

phacurcas

L.Jatro

phacurcas

L.endo

phyte

6.8

70.8

6255

⋅⋅⋅

Temasek

Lifesciences

Labo

ratory

Methylobacterium

sp.

EUR3

AL-11

GCA

000526475.1

—thermaladaptio

nand

carbon

metabolism

inperm

afrost

7.21

71.1

6670

⋅⋅⋅

DOEJointG

enom

eInstitute

Methylobacterium

sp.

UNCC

L110

GCA

000745415.1

—bacteria-plant

association

6.61

69.7

—⋅⋅⋅

DOEJointG

enom

eInstitute


nodule formation [18, 19], while other Methylobacteriumspecies may be related to phytohormone production [20] orinteracting with plant pathogens [17, 21], promoting plantgrowth [22, 23] and inducing higher photosynthetic activity[24].

AlthoughMethylobacterium spp. are not phytopathogenicbacteria and are not associated with the degradation of plantbiomass (saprophytes), it was reported that some strains areable to synthesize pectinase and cellulase, suggesting theycan induce systemic resistance during plant colonization[22, 25]. In addition, they can help plant development bydecreasing environmental stress and by degrading toxicorganic compounds [26], immobilizing heavy metals [27],or even inhibiting plant pathogens [28]. Thus, these bacteriacan play an important role in the microbial balance in plants,highlighting their importance during plant development.

Therefore, this review aims to give an overview ofreported genes and proteins involved in Methylobacterium-plant interaction process, not only the mechanisms thatinduce plant growth, systemic resistance, and plant pathogeninhibition but also involved in the bioremediation of organic(herbicides) and inorganic (heavy metals) compounds fromcontaminated crop soil or water that can be used to irrigatethe agriculture soil. The present review also comprises thestudies that use genomic, transcriptomic, proteomic, andmetabolomics to identify the genes and proteins potentiallyinvolved in this plant interaction.The comprehension of howthis Methylobacterium-plant interaction occurs can allowus to increase crop production and decrease environmen-tal pollution, possibly creating a biotechnological product.Therefore, besides an agronomic application, other biotech-nological uses of this genus will also be reported duringthis review showing the potential of these facultative pink-pigmented methylotrophic genera.

2. Methylobacterium duringPlant Interactions

Bacteria may interact with plants, acting as pathogens, epi-phytes or endophytes. Endophytic microorganisms aredefined as those which live part of or whole life insidethe plant tissues without causing damage or forming visibleexternal structures to the host [29, 30]. Thus, this defini-tion excludes mycorrhizal fungi, nodulating symbiotic bac-teria, epiphytic microorganisms, and phytopathogens [31].Depending on environmental conditions, a microorganismclassified as an endophyte can behave like an epiphyte or asubclinical pathogen [32]; in some cases, the presence of anendophyte can be associated with the presence of a pathogenor another microorganism, between which there may be anintimate interaction [21].

Methylobacterium spp. can be found in association withmore than 70 species of plants [16], actively colonizing theroot, branches and leaves [12] with several studies report-ing Methylobacterium as a putative endophyte of differenthost plants, such as cotton [7], peanut [22], citrus [21],pinus [33], eucalyptus [34], sunn hemp [18], Catharanthusroseus, tobacco [12], strawberry [35], and mangrove plants[27]. Knief et al. [36] reported based on the metagenomics

and metaproteomics approach that alphaproteobacteriumare found more frequently in rice phylosphere than in ricerizosphere, where the most abundant genera are RhizobiumandMethylobacterium indicating that these genera prefer thehost plant environment. Furthermore, within this genus thereis a diversity ofMethylobacterium species inside the plant host[21, 34].

The first step of the plant-bacteria interaction is therecognition of plant exudates by the bacteria. Such exudatesare composed mainly of sugars, amino acids, and organicacids as well as flavonoids [37, 38], and they are able toattract specific and beneficial microorganisms [39], establish-ing an indwelling bacteria-plant interaction. Root exudatespossibly influence host recognition, biofilm formation, andcolonization by Methylobacterium spp. as endophytes [13].After plant recognition, surface colonization likely dependson traits such as adhesins, pili, and EPS (exopolysaccharides)to attach to the cells on the surface. Kwak et al. [40]reported the presence of ten genes involved in type-IVpilus biosynthesis and two genes related to hemolysin-typeadhesins in M. oryzae CBMB20. Numerous studies havereportedMethylobacterium spp. cells colonizing plant tissuesin a mucilaginous layer [12, 13, 41], suggesting that this couldbe a first step during plant colonization.

Thus, the endophytic bacteria are able to penetrate intothe plant, generally through root wounds, and systemicallycolonize the host, inhabiting the apoplast [42], conductivevessels [42, 43] and occasionally the intercellular space[44]. However, colonization is somehow guided towardplant-bacteria-specific interactions. Poonguzhali et al. [41]observed intercellular colonization in tomato roots by M.suomiense CBMB120, but not in rice. Through systemiccolonization, these bacteria can change the physiologicaland morphological conditions of the host, and they canaffect the populations of other microorganisms present inthe plant [12]. Reinhold-Hurek and Hurek [30] reportedthat endophytic bacteria may influence the physiology ofthe host plant by processes that are not yet clarified. Thisinfluence is due to the close relationship between the differentindividuals who developed during the coevolution of species.Bacteria present several properties of interest within theirhosts, protecting them from insect attacks, pathogens, andherbivores, producing plant growth hormones, and providingmore nutrients and biological nitrogen fixation [45].

Plant colonization by the endophytes may be guided bysome quorum sense (QS) systems, using signaling moleculescommonly found in Gram-negative bacteria, such as N-acyl-homoserine lactones (AHLs), which are regulated by theLuxI/LuxR system [46]. Bacteria can work as a multicellularorganism due to the QS system once the bacterial popu-lation growth and the extracellular concentration of AHLsincreases, reaching a level that can regulate the transcriptionof different genes that may be related to the secretion system,biofilm formation, exchanges ofDNAand others [47].Methy-lobacterium strains produce AHL molecules [48], which areresponsible for bacterial cell-to-cell communication [43] andare produced with an increase in bacterial cell density. Thismolecule, in Methylobacterium sp. strain GXF4, may influ-ence bacterial communication and colonization outcomes


within the xylem [43], indicating that Methylobacteriumstrains may also interact with other microorganisms insidethe plant, including phytopathogens such as Xylella fastidiosa[49]. M. mesophilicum SR1.6/6, isolated from the interior ofthe sweet orange tree (Citrus sinensis) [21], is also able to pro-duce six long-chain homoserine lactones (HSLs) (the satu-rated homologs (S)-N-dodecanoyl and (S)-N-tetradecanoyl-HSL, the uncommon odd-chainN-tridecanoyl-HSL, the newnatural product (S)-N-(2E)-dodecenoyl-HSL, and the rareunsaturated homologs (S)-N-(7Z)-tetradecenoyl, and (S)-N-(2E,7Z)-tetradecadienyl-HSL), as described by Pomini et al.[48]. For this strain, the (S)-N-dodecanoyl-HSL moleculewas able to upregulate the expression of genes related toplant-bacteria interactions, such as bacterial metabolism(mxaF), adaptation to stressful environments (crtI and sss),interactions with plant metabolism compounds (acdS) andpathogenicity (patatin), showing that the AHL molecule,together with bacterial density, activates several plant-bacteria interaction genes [50]. These results imply that,althoughMethylobacterium spp. are able to identify the plantexudate and trigger a response, during plant colonization,the gene coordination may also be regulated by a quorum-sensing system to allow efficient plant colonization.

2.1. The Role of Methylotrophic Metabolism during Plant Inter-actions. Methylotrophic bacteria, including Methylobacte-rium spp., were first reported by Anthony [51].These bacteriaare able to use (C1) compounds (mainly methanol but alsoformaldehyde and formate [7]) as a sole carbon source,or they can use multicarbon compounds with or withoutcarbon-carbon bonds. Methylobacterium spp. can also useother carbon sources: those with two carbons, such as acetate,ethanol, and ethylamine; those with three carbons, such aspyruvate; or those with four carbons, such as succinate [52].This ability to use several carbon sources allowsMethylobac-terium spp. to colonize different environments, includingplants, which release methanol by stomata during plantgrowth [35].

Bacterial methylotrophic metabolism starts in the peri-plasm, where the methanol dehydrogenase (MDH) enzymeoxidizes methanol into formaldehyde (Figure 2). MDH is an𝛼2𝛽2tetramer with two active sites, a prosthetic group and a

calcium atom [5]. The large and small subunits are encodedby the mxaF and mxaI genes, respectively; moreover, mxaGencodes cytochrome c, the primary electron acceptor forMDH [53]. MDH is composed of two large (66 kDa) and twosmall (8.5 kDa) subunits. The large subunit (MxaF) is essen-tial for methanol dehydrogenase activity; it contains a PQQprosthetic group [54]. This enzyme is a soluble quinoproteinthat uses pyrroloquinoline quinone (PQQ) as a cofactorto transfer two electrons to cytochrome c [53]. Methanoloxidation generates formaldehyde (a main intermediate ofmethylotrophic metabolism), which can also be assimilatedin the serine cycle and used by the cell or oxidated to CO

2,

generating energy; each molecule of methanol generates 1ATP [54].

Chistoserdova et al. [55] observed that M. extorquensstrain AM1 contains 70 genes that are related to methy-lotrophic metabolism. These genes are distributed into eight

regions of the bacterial chromosome. One 12.5 kb clus-ter contains 14 genes transcribed in the same direction(mxaFJGIRSACKLDEHB), and upstreamof this cluster thereis the mxaW gene transcribed in the opposite direction.There are also the transcriptional regulators mxbMD andmxcQE [55]. Moreover, the M. extorquens AM1 genomecontains two copies of the xoxF gene, which encodes a PQQ-dependent periplasmic alcohol dehydrogenase that is 50%identical to mxaF. When both xoxF genes are absent, thestrain lacks methanol dehydrogenase activity and losses boththe ability to grow in methanol as the sole carbon sourceand the mxa promoter, suggesting that xoxF is responsiblefor the regulatory complex [54] that influences methanolmetabolism.

The major source of methanol in the atmosphere is for-ests, due to plant emissions [56] because during plant growth,cell expansion depends on pectin breakdown by pectinmethylesterase, resulting in the production of methanolthat is released by stomata [35]. Methanol production mayfluctuate according to environmental conditions, such asflooding [57], plant age [52], and physiological state becausein mature (yellow) leaves and during abscission, methanolrelease increases significantly [58]. In this way, during plantcolonization, Methylobacterium spp. may take advantage ofthe presence of methanol released by plants by expressinggenes related tomethylotrophy [59], such asmxaF, colonizingthe host plant more efficiently than other plant-associatedbacteria. In fact,mxaF-defectiveM. extorquensmutants wereless competitive than the wild-type strain during the col-onization of Medicago truncatula under competitive con-ditions [14]. The authors observed that the ability to usemethanol as a carbon and energy source provided a selectiveadvantage during host colonization. However, after a singlecolonization the defective mutants were able to colonizethe plant tissues, suggesting that methanol is not the onlycarbon source that is used by Methylobacterium spp. duringendophytic and epiphytic plant colonization.

2.2. Induction of Plant Growth. The cascade of events thatoccurs after a bacterial cell recognizes plant exudates resultsin major changes in cellular metabolism, including the accu-mulation of several secondary metabolites [45]. Such physio-logical changes can modulate the growth and developmentof the plant. However, considering their complexity, thesemechanisms and networks are still far from being elucidated.Therefore, different studies have been conductedwith the aimof uncovering these mechanisms.

Plant growth stimulation by endophytic bacteria is largelydue to phytohormone production, and several studies havebeen reported the interaction of Methylobacterium specieswith different plant species by regulating phytohormone pro-duction [60]. Methylobacterium strains have been reportedto produce phytohormones such as cytokinins and auxins[61], which promote cell division and elongation, respectively.Pirttila et al. [62] tested for the most common gibberellinproduction in M. extorquens, but such compounds were notfound. Instead, the bacterium produced adenine derivativesthat may be used as precursors in cytokinin biosynthesis. Ina recent study, Kwak et al. [40] showed that the M. oryzae


O

HN

OH

O

HN

OH

Degrade toxiccompound

ACC ACC

SAM

Ethylene

Plant stress

growth

DCM

Formaldehyde

Methanol

Siderophore production

Plant growth promotion

P solubilization

available to plants

ISR Glucose Carbon

source

Enzyme production

Cl

Cl

HH

H

HC C

O

H HH

H

OH

OHOHOH

HO

CH2OH

Phosphatase

mxaf

dcmA

MethylobacteriumPlant root

AuxinAuxin

Nitrogen

fixation and

nodulation

Fe++

Fe++

Fe++

Cd++

Cd++

Pectin + cellulose +

Cell wall

hemicellulose

Cytokines

N 2

NH4+

HPO42− HPO4

2−

Cytokinins

ACC deaminase

𝛼-ketobutyrate +NH3

CaPO4

Cellulase

Pectinase

HPO42−

CH3OH

CH3OH

CH3OHCH3OH

CH3OHCH3OH

CH3OH

CH3OHCH3OH

CH3OH

CH3OH

↓ Pathogen

↑ Plant tolerance

Figure 2: Molecular mechanisms possibly involved in plant colonization and plant growth promotion identified in Methylobacterium spp.genomes. The molecular communication during plant-Methylobacterium species interaction involves bacterial proteins and the secretion ofphytohormones (auxin and cytokinin) that induce plant growth and decrease pathogen growth.Methylobacterium spp. are able to modulateethylene levels using ACC (an ethylene precursor) as a source of nitrogen. The auxin and ethylene pathways are related, whereas ACC plantproduction is induced by bacterial auxin. Methylobacterium spp. can also solubilize phosphorus and produce siderophores that can chelateiron and other metals recognized and absorbed by the plant, increasing nutrient uptake (mainly Fe). The bioremediation of toxic organiccompounds is also be observed during DCM degradation, as is the chelation of inorganic toxic compounds, increasing plant tolerance.Methylotrophic metabolism bacteria present an adaptive advantage due to methanol exudation by plant leaves. These molecular processesmodulateMethylobacterium spp. plant colonization and plant defense.

CBMB20 features two miaA genes, which are critical for theproduction of zeatin, a major cytokinin. Another importanthormone is auxin.Themain auxin in plants is indole-3-aceticacid (IAA), which controls an important physiological pro-cess during root development [63]. In the Methylobacteriumgenus, genes that encode enzymes related to auxin biosyn-thesis, such as amine oxidase, aldehyde dehydrogenase, nitri-lase/cyanide hydratase, N-acyltransferase, nitrile hydratase,amidase, have been reported [22, 23, 40].Methylobacterium isable to produce IAA [64], suggesting that its inoculation canincrease plant IAA concentrations and induce plant growthpromotion [65].

Another compound that regulates root growth and devel-opment is ethylene [66], which is related to the auxinbiosynthesis pathway [39]. High concentrations of ethylene

are related to stress conditions in plants and may have adeleterious effect on plant growth, inhibiting root elongationand accelerating abscission, aging, and senescence [67]. Inethylene biosynthesis, the precursor of the ethylene hormoneis ACC (aminocyclopropane-1-carboxylic acid), which isconverted from S-adenosylmethionine (SAM) to ethylene bythe actions of ACC synthase (ACS) and ACC oxidase (ACO),enzymes that are transcriptionally regulated independentlyby both biotic and abiotic factors [39, 66, 67]. Plant ACSactivity can also be increased by bacterial IAA production,showing that both pathways are related, and may increaseplant and bacterial fitness during Methylobacterium-plantinteractions (Figure 2). There are also suggestions that theamount of IAA released may have an important role in mod-ulating plant-microbe interactions, and the balance between


IAA and ethylene might be fundamental for the maintenanceof endophytic bacterial plant colonization, as proposed byHardoim et al. [39].

Moreover, endophytes carry several important genesrelated to beneficial interactions with the host plants, includ-ing the acdS gene [39]. The acdS gene encodes an ACCdeaminase enzyme that converts ACC into ammonia (NH

3)

and 𝛼-ketobutyrate. An analysis of the genomes of Methy-lobacterium species revealed that the plant-associated species,such asM. oryzae,M. nodulans, andM. radiotolerans, containthis ACC deaminase gene [40] and thatM. nodulans andM.radiotolerans are able to use ACC as a nitrogen source by theactions of ACC deaminase, reducing ethylene levels [60] andconsequently the stress ethylene response in the host plant.Joe et al. [68] reported that the association between an ACCdeaminase-positive M. oryzae CBMB20 strain with Azospir-illum brasilense CW903 strain reduced ethylene levels inplants. These authors developed coaggregated cell inoculantscontaining both strains, which improved plant resistance andreduced stress in inoculated tomato plants. Similar resultswere observed in canola: when a plant-growth promotingMethylobacterium containing ACC deaminase was inocu-lated into canola roots, it also reduced the concentrations ofACC and ethylene in the plant, increasing root length [65,66].Therefore, bacteria that are able to reduce ethylene levelsin the host plant are associated with plant growth promotion[39, 60, 66].

In addition to phytohormone production, Methylobac-terium presents other beneficial plant interactions, improvingthe cycling of nutrients such as siderophore production,which is important to increase the iron supply to the plantand to reduce heavy metal toxicity [69]; nitrogen fixation,which favors plant biomass increase [18, 70] and phosphatesolubilization, making phosphate available for plant uptake[71]. All of these processes are considered to be involved inplant nutrient acquisition and are responsible for promotingplant growth.

Nitrogen is often the most limiting nutrient for plantgrowth, but nitrogen from the atmosphere is unavailableto plant metabolism. Thus, the process of nitrogen fixationinvolves the transformation of atmospheric nitrogen intoammonia, which is available for plant use. The biologicalreduction of nitrogen to ammonia can be performed onlyby some prokaryotes with the presence of the nitrogenaseenzyme [19]. M. nodulans was originally isolated from Cro-talaria podocarpa [18] and together with Methylobacteriumsp. 4-46 represents the few nodulating Methylobacteriumspecies reported so far [40]. M. nodulans ORS2060 wasreported to contain the nifH gene (involved in nitrogenfixation) and to induce nitrogen-fixing nodules on the rootsof legumes by the nodA gene [15]. Nodules are specializedorgans in which fixing nitrogen bacteria reduce atmosphericnitrogen to ammonia [18]. M. nodulans seems to have acompetitive advantage during plant colonization and nod-ule formation because of its ability to obtain carbon bothfrom sugars (host plant photosynthesis) and methanol (frommethylotrophy) [72]. It was previously reported that the lossof the bacterialmethylotrophic function significantly affectedplant development because the inoculation of M. nodulans

nonmethylotrophic mutants in C. podocarpa decreased thetotal root nodule number per plant and the whole-plantnitrogen fixation capacity, also reducing the total dry plantbiomass compared with the wild-type strain [70].

Another essential nutrient present in the soil is phos-phorus. Despite the high concentrations of total phosphor insoils, most of it is in the form of inorganic compounds boundto calcium, iron, or aluminum or immobilized in organicmatter such as phytate (phytic acid,myo-inositol hexakispho-sphate), the most abundant organic phosphorus compoundin soil [73]; therefore, it is not available for plant uptake[74]. Single-cell organisms assimilate mainly soluble ionicphosphate forms (HPO

4

2−, H2PO4

−), but the concentrationof soluble phosphorus in soil is usually very low. There areconsiderable populations of phosphate-solubilizing bacteriain soil and in plant rhizospheres, which are important forincreasing plant biomass by converting both organic andinorganic insoluble phosphate to a form available to plants[75]. Among those,Methylobacterium spp. have the ability todissolve inorganic phosphates, whichmay be further involvedin phosphate metabolism in both microorganisms and plants[76].There are three different types ofmicrobial enzymes thatsolubilize organic phosphate: nonspecific acid phosphatase,phytase and C-P lyase (or phosphonatase). They all releasephosphate, the first from phosphoric ester or phosphoricanhydride, the second from phytic acid, and the third fromorganophosphonates. M. oryzae has been reported to havegenes encoding all three phosphatase enzymes [40].

Another positive plant-bacteria interaction attribute isthe ability to synthesize bacterial siderophores. Siderophoresare low-molecular-mass compounds with a high affinity foriron that are produced by bacteria to solubilize iron topromote its efficient uptake. Iron is extremely necessary toalmost all forms of life because it participates in numerousbiological processes; however, it exists mainly as insolubleFe3+ in aerobic environments [77]. Therefore, siderophorerelease is an effective strategy developed by bacteria for ironacquisition that can, in turn, make this metal also availablefor plant uptake, contributing to plant growth [78]. In theMethylobacterium genus, the iron uptake genes iucAand iucChave been described in 35 strains, including M. extorquensstrains AM1, PA1, DM4, and CM4 andM. populi [23].

In a study usingWC-MS (whole-cell matrix-assisted laserdesorption ionization-time of flight mass spectrometry),siderophore production and phosphate solubilization wereanalyzed in 190 unique strains of Methylobacterium speciescollected from leaf samples of many host plants [79]. Amongthese Methylobacterium isolates, 185 (growing on methanolas a carbon source) and 93 (growing on glucose as a carbonsource) strains were positive for calcium phosphate solubi-lization. Siderophore production was positive in 35 strains[79]. Lacava et al. [80] also evaluated siderophore productionby 37 Methylobacterium spp. strains and observed that allthe tested strains were able to produce hydroxamate-likesiderophores, but not catechol-like siderophores, suggestingthat these strains are able to bind specific metals [77].


2.3. Inhibition of Plant Pathogens. In recent decades, inter-action studies have shown that the presence of endo-phytic microorganisms can increase plant protection againstpathogen attacks [81, 82]. Thus, despite of the presence oflarge numbers of potential phytopathogenic microorganismsinside the plant, most of these interactions remain asymp-tomatic, due to an elaborate plant defense system [83, 84] andthe stability of the microbial community.

Endophytes, including Methylobacterium spp., can pro-tect host plants by the synthesis of a large spectrum of antimi-crobial molecules [85], nutrient competition with pathogens[86, 87] or by inducing systemic resistance (ISR, InducedSystemic Resistance) [88]. ISR can be induced by volatileorganic compounds released from some bacteria [89] andby genes of bacteria that encode plant cell wall degradationenzymes such as glycosidases, cellulases (or endoglucanase)and hemicellulases [90] and pectinase [22, 25] (Figure 2).This mechanism (ISR) has also been reported to induce plantgrowth and to protect plants against pathogens [22, 81, 91].Ardanov et al. [92] observed that even at a low density,a Methylobacterium sp. IMBG290 inoculum was able toinduce potato resistance against Pectobacterium atrosepticumby activating the plant antioxidant system; however, at ahigh density this positive effect was not observed, resultingin susceptibility to the pathogen. In a more recent study,Ardanov et al. [81] evaluated the ability ofMethylobacteriumsp. IMBG290 to induce resistance in several potato (Solanumtuberosum L.) cultivars against P. atrosepticum, Phytophthorainfestans, and Pseudomonas syringae pv. tomato DC3000, aswell as M. extorquens DSM13060 in pine (Pinus sylvestrisL.) against Gremmeniella abietina. In addition, the authorsobserved that plants inoculated with Methylobacterium spp.and challenged with the pathogen presented a differentendophytic community compared with uninoculated controlplants, suggesting that endophyte inoculation may have aneffect not only on pathogen establishment but also on plantmicrobial communities.

Yim et al. [91] reported the induction of defense responsesin tomato challenged with Ralstonia solanacearum aftertreatment with four differentMethylobacterium strains. Theyverified a reduction in ACC accumulation and consequentreductions in ethylene levels and disease symptoms. In addi-tion, the authors observed an increase in defense enzyme con-tents, suggesting the potential use of methylotrophic bacteriaas biocontrol agents in tomato crops. Furthermore, seedtreatment with Methylobacterium sp. induced significantprotection against Aspergillus niger and Sclerotium rolfsiipathogens in groundnut under pot-culture conditions [22].Furthermore, the biocontrol potential of Methylobacteriumspp. for fungal pathogens such as Fusarium udum, F. oxys-porum, Pythium aphanidermatum, and Sclerotium rolfsii wasalso reported under in vitro conditions [28]. Kwak et al.[40] described the presence of several genes in M. oryzaeCBMB20 involved in antimicrobial compound production,such as bacteriocin and 4-hydroxybenzoate as well as 536genes related to transport, such as amino acid and saccharidetransporters, porins, the major facilitator superfamily ofpermeases, the RND family of efflux transporterMFP subunitproteins, and urea ABC transporters.

In citrus, it was observed that the Methylobacteriumgenus is dominant within branches. Considering the isolationfrequency variation, it has been suggested that Methylobac-terium spp. interact with Xylella fastidiosa, the causal agentof CVC (citrus variegated chlorosis) [17, 21]. The presenceof the endophytic bacterium M. mesophilicum in internaltissues of asymptomatic citrus plants (hosting X. fastidiosa)could stimulate the production of compounds that promotethe resistance of these plants to X. fastidiosa or reduce thegrowth of this vascular phytopathogen [17], limiting theestablishment ofX. fastidiosa in asymptomatic plants. In vitrointeraction studies revealed that M. mesophilicum SR1.6/6inhibits the growth of X. fastidiosa, while M. extorquensAR1.6/2 has no effect on this pathogen [17]. This result wasconfirmed by Lacava et al. [49], who observed a lower pop-ulation of M. mesophilicum SR1.6/6 and X. fastidiosa 9a5c inCatharanthus roseus when these bacteria were coinoculated,suggesting that these endophytic and pathogenic bacteriacould compete for nutrient and space inside the host plants.

This competitionmay occur both inside the xylem vesselsand inside the insect vectors. Methylobacterium spp. plantcolonization begins with biofilm formation on roots [13];the bacteria then colonize the internal tissues of the hostplant, including xylem vessels [93]. From the xylem vesselsof the host, this endophytic bacterium may be transmit-ted from plant to plant by sharpshooter vectors, such asBucephalogonia xanthophis [94]. This insect vector is alsoable to transfer X. fastidiosa from infected plants to healthyplants [95], suggesting that these bacteria may interact indifferent ways inside hosts. This opens a field to searchfor new strategies for the symbiotic control of pathogens,paratransgenesis. Paratransgenesis means the genetic alter-ation of symbiotic microbes that are carried by insects tocompete with pathogens and control their colonization [94].Because M. mesophilicum was identified between bacterialpopulations naturally associatedwith themain sharpshootersresponsible for the transmission of X. fastidiosa [96], speciesof the Methylobacterium genus have been a promising targetfor such engineering [93].

In addition, studies that aim to investigate the genesinvolved in plant-bacteria interactions may expand theunderstanding ofMethylobacterium-plant-pathogen interac-tions and help assess whether there is bacterial genotypespecificity to host plants [6, 97]. However, further studies areneeded to better understand the molecular and biochemicalmechanisms involved in these interaction processes. Ulti-mately, the discovery of biocontrol agents is the main goalin the search for the reduction of pesticides in agriculture,which may have a negative impact on human health and theenvironment.

3. Bioremediation UsingMethylobacterium spp.

Methylobacterium spp. are able to biodegrade a variety oforganic toxic compounds according to several reportedstudies. Van Aken et al. [98] observed thatMethylobacteriumsp. strain BJ001 in pure culture was able to degrade sev-eral toxic explosives, such as 2,4,6-trinitrotoluene (TNT),


hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX) and octahy-dro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX), in 10 to 55days. M. extorquens DM4 also has the ability to degradea volatile and toxic halogenated solvent, dichloromethane(DCM, CH

2Cl2), which is mainly used and produced by

industry [26]. This process occurs by converting DCM intoformaldehyde, an intermediate of methylotrophic metabolicgrowth [99] (Figure 2). The industrial degradation of for-maldehyde was reported and tests were performed in a bio-reactor, showing that Methylobacterium sp. XJLW presentstolerance to 60 g⋅L−1 of formaldehyde and 1,687.5mg⋅L−1⋅h−1of degradation rate, being able to degrade 5 g⋅L−1 of formalde-hyde [100]. Phenol degradation was also observed. Indus-trial wastewater can contain high concentrations of phenol;Khongkhaem et al. [101] reported that Methylobacterium sp.NP3 and Acinetobacter sp. PK1 encapsulated in silica-treatedphenol (7500 to 10000mg⋅L−1) contaminatedwater efficientlyfor up to 55 days.

In industrial areas, there are several soil contaminants.In a former industrial site in Italy, the main problem waspolycyclic aromatic hydrocarbons (PAHs), a toxic compoundresulting from industrial treatment and waste combustionthat was present in the studied area. Ventorino et al. [102]showed that M. populi VP2, which has several character-istics that promote plant growth, was the isolate best ableto degrade PAHs. Another common soil and groundwa-ter pollutant is MTBE (methyl-tert-butyl ether), which iswidely used as fuel. It was reported that a reactor able totreat tap water presenting methylotrophic bacteria, includingMethylobacterium spp., showed a high (over 99.9%) efficiencyof MtBE degradation [103]. TCE (trichloroethylene), whichis a solvent to remove grease from metal parts, is also asoil pollutant. A combination of a few bacteria, includingMethylobacterium spp., was able to degrade MTBE and TCEin the presence of heavy metals at a high efficiency (49–182% higher than noninoculated) [104]. These capabilitiessuggest that bacteria from this genus may be used for thebioremediation of contaminated environments, such as soiland water.

The increasing use of chemical fertilizers and pesticides,results in an increase in heavy metal concentrations in soil,leading to a great environmental impact [105]. Unlike organiccontaminants, metals are not degradable and remain in theenvironment for long periods of time; when present at highconcentrations in soil, metals can negatively affect plantmetabolism, reducing plant growth [106, 107]. Therefore,a tolerant microorganism can help to bioremediate con-taminated water by flocculation [108] and soils by heavymetal immobilization [109], leading to an increase in planttolerance or even increasing phytoextraction [110] duringbacteria-plant interactions. The potential of the methy-lotrophic genera is shown by their tolerance to high dosesof several heavy metals, such as nickel (Ni), cadmium (Cd),cobalt (Co), zinc (Zn), chrome (Cr) [111], arsenic (As), lead(Pb) [27], and mercury (Hg) [104]. Moreover,M. oryzae wasable to increase Cd and Ni tolerance in tomato plants bydecreasing Cd and Ni uptake and promoting plant growth[107].

Haferburg and Kothe [112] reported four main tolerancemechanisms to heavymetals in bacteria: (i) biosorption—themetal binds to the bacterial cell wall, becoming unavailable;(ii) intracellular sequestration—the metal is chelated to acompound inside of the cell; (iii) efflux transporter—themetal is expelled from the cell by a membrane pump [113];and (iv) extracellular chelation—a chelating compound ispumped out of a bacterial cell, complexing the metal andmaking it unavailable to other living organisms. An exampleis the siderophore molecule [114, 115] (previously mentionedin “Section 2.2”), which is able to chelate iron, making itunavailable to pathogens but available to plants. Streptomycestenda F4 was reported to produce siderophores that are ableto bind to Cd, decreasing Cd availability to other organismsand decreasing soil availability [114].

Genes related to heavy metal tolerance (uptake andefflux) have been reported in M. oryzae, including genesrelated to the cation efflux system protein CzcA, which areinvolved in cobalt-zinc-cadmium resistance; several ABCtransporters involved in zinc and nickel uptake; copper-translocating P-type ATPase involved in copper resistance;and genes that encode arsenic/arsenate resistance and chro-mate transport protein. Other transport and secretion sys-tems reported in the M. oryzae genome may be relatedto metal tolerance, for example, the RND family proteinsresponsible for efflux transport [40].

Therefore, in addition to increasing plant growth andinhibiting plant pathogens, this genus has been reportedto increase plant tolerance to heavy metals and to degradetoxic organic compounds in soil, decreasing in this way plantstresses and benefiting evenmore bacteria-plant interactions.In addition, this genus is able to produce biodegradableplastic and other industrial products, showing its greatplasticity and its agricultural and industrial importance.

4. Biotechnological Uses ofMethylobacterium spp.

Methylotrophic bacteria, in addition to playing a key rolein bioremediating contaminated environment and in plantgrowth, can produce several industrial products and biode-gradable compounds. Petroleum-based plastic has a highhalf-life, causing several problems to the environment. Somebacteria, including Methylobacterium spp., are able to pro-duce highly resistant biodegradable plastic that is similarto conventional plastic. Examples of such biopolymers arethe biodegradable polyesters polyhydroxybutyric acid (PHB)and polyhydroxyalkanoate (PHA).M. extorquenswas geneti-cally modified to increase PHB and PHA production usingmethanol as a substrate [116]. M. organophilum was alsoreported to accumulate PHB and PHA [117] under nitrogenlimitation while biodegrading methane (a greenhouse gas).Other studies reported that the production of PHB inMethylobacterium sp. GW2 could reach 40% w/w of bacterialdry biomass; when supplemented with valeric acid, theyalso produced the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) [117]. In addition,Methylobac-terium sp. ZP24 enhanced PHB production when usingprocessed cheese (supplemented with ammonium sulfate)


instead of lactose or sucrose in a feed batch [118], and M.extorquens AM1 was recently reported to accumulate poly-hydroxyalkanoate (PHA) copolymers under cobalt-deficientconditions [119].

Genetic approaches involving PHB biosynthesis wereperformed, showing that phaA, phaB, and phaC encodingbeta-ketothiolase, NADPH-linked acetoacetyl coenzyme A(acetyl-CoA) reductase, and PHB synthase, respectively, werepresent in theM. extorquensAM1 genome and are responsiblefor PHB synthesis. Furthermore, the authors reported thata phaB mutant was not able to grow in methanol, showingthat PHB synthesis genes also affect growth in C

1and

C2compounds in the methylotrophic M. extorquens AM1

[120]. In a second study, three more genes, gap11, gap20(which encode phasins, granule-associated proteins), andphaR (which controls acetyl-CoA flux), were identified to beinvolved in PHB biosynthesis [121].

Another compound that Methylobacterium spp. are ableto produce is glyoxylate, an important compound in perfumemanufacture and an intermediate in drug and pesticideproduction [122]. Using genetic engineering, Shen and Wu[122] made a strain able to overexpress the hydroxypyruvatereductase enzyme, a key component in the serine cycle,leading to glyoxylate accumulation.

In agriculture, Methylobacterium spp. can contribute toseveral biotechnological applications. Of all the beneficialcharacteristics during plant interactions reported above (in“Section 2.2”), Polacco and Holland in 1991 patented (PatentnumberUS5268171) a process that usedmethanol to selectM.mesophilicum in the plant and to alter plant metabolism, pro-moting plant growth. In 1995, Holland and Polacco depositedanother patent (Patent number US5512069), in which theycoated or impregnated seeds with at least one PPFM toimprove seed germination, affirming that PPFM can be usedto produce cytokinin. Verginer et al. [123] reported thatM. extorquens DSM 21961 in vitro can increase the produc-tion of two furanoid compounds, 2,5-dimethyl-4-hydroxy-2H-furanone (DMHF) and 2,5-dimethyl-4-methoxy-2H-furanone, which are responsible for strawberry flavor, show-ing that the bacteriumcan influence fruit quality.Nasopoulouet al. [124] reinforced that hypothesis, showing that theexpression of the alcohol dehydrogenase enzyme by endo-phytic bacteria and the flavor components (DMHF) were inthe same tissues.

Genetic engineering studies were also performed withMethylobacterium spp. A cryAa gene that encodes a proteinwith activity against lepidoptera from Bacillus thuringien-sis was cloned and expressed in M. extorquens, using themxaF (MDH) promoter, expressing the recombinant Cry1Aaprotein and obtaining crystals similar to those formed bythe original organism, B. thuringiensis, suggesting that thisorganism could be used to promote plant growth (naturally)and inhibit crop pests (due the transgenic gene) [125]. Ina similar way, the 𝛽-1,4-endoglucanase A gene (encodingEglA) from Bacillus pumilus was expressed in M. extorquensAR1.6/2 [93], allowing this strain to present cellulolyticactivity (ranging from 0.73 to 1.16U⋅mL−1) [126] and tocolonize the plant xylem without inducing disease symptoms

in the host plant. This M. extorquens AR1.6/2 strain wasisolated from the inner tissues of citrus plants infected withXylella fastidiosa, suggesting that this genetically modifiedMethylobacterium species could be used as an agent ofsymbiotic control [93] because this endoglucanase coulddegrade a gum produced by X. fastidiosa.

5. Omics Studies ofthe Methylobacterium Genus

The advancement of molecular biology and the increas-ing use of next-generation sequencing have enabled thesequencing of many bacterial genomes. Until 2012, only 11Methylobacterium genomes were available. Currently, thereare 22 sequenced genomes of the Methylobacterium genusdeposited in the National Center for Biotechnology Infor-mation (NCBI) database: M. extorquens (AM1, DM4, PA1,DSM13060, CM4); M. nodulans ORS2060; M. populi BJ001;M. radiotolerans JCM2831; M. mesophilicum SR1.6/6; and 13uncharacterized strains (Methylobacterium spp.: 4-46, GXF4,MB200, 77, WSM2598, 285MFTsu5.1, 10, B1, B34, 88A, L2-4,EUR3 AL-11 and UNCCL110). The GXF4 strain was the firstgenome announcement of a plant xylem-associated strainof the Methylobacterium genus [43], and SR1.6/6 was thefirst genome announcement of a Methylobacterium strainassociated with a tropical plant [127] (Table 2).

Overall, genomes deposited in NCBI have been isolatedfrom different sources, such as air, biogas reactors, plants,and contaminated soils and lakes, showing that the greatphenotypic plasticity of those genomes allows the colo-nization of different niches. Methylobacterium genomes arecharacterized by a GC content between 66.7 and 71.5%, agenome size between 4.6–7.8Mbp and a plasmid numberbetween 2 and 8 (there is little information about plasmidsin the literature) (Table 1). Normally, plasmids encodemainlyproteins of unknown function or proteins associated withplasmid-related functions, such as genes for antibiotic resis-tance and genes for virulence. However, in the AM1 strainsome different plasmid functions were reported, such asgenes related to cation efflux systems, a cluster of copperresistance genes, a truncated luxI gene and UmuDC systems[128].There is also an approximately 130 kb region in the AM1megaplasmid that is syntenic to a region of similar lengthin the chromosome of strain DM4. In the CBMB20 strainplasmid, genes encoding DNA polymerase V subunits C andD were observed [40].

Several of these sequenced genomes ofMethylobacteriumstrains have been isolated from plants or reported to interactwith plants: PA1, DSM13060, ORS2060, BJ001, AM1, GXF4,B1, B34, L2-4, and SR1.6/6, from which four strains wereisolated endophytically: SR1.6/6, BJ001, GXF4, and L2-4.Each strain presents a specific interaction with the hostplant, and successful colonization may vary according tothe species and the stage of plant development [6, 34]. Thisdifference in the success of plant colonization can also beassociated with its genome because each strain presentsdifferent sizes and numbers of replicons, as well as a specificset of genes, possibly with unknown functions for each strain.The colonization and distribution in the host can also be


Table 2: Omics studies in theMethylobacterium genus.

Organism Approach Findings References

M. extorquens AM1 Proteome Identify a PhyR stress regulator during phyllosphere colonizationusing 2D analysis.

[59]

M. extorquens AM1 ProteomeProteomic comparison under single carbon (methanol) andmulticarbon (succinate) growth in a gel free quantitative proteomicassay.

[134]

M. extorquens AM1 Proteome Cytosolic protein differentially modulated under single carbon(methanol) and multicarbon (succinate) growth.

[135]

M. extorquens AM1 ProteomeCompare wild type withM. extorquens AM1 lacking isocitratelyase (the key enzyme in the glyoxylate cycle) during growth onacetate, which was replaced by ethylmalonyl-CoA pathway.

[138]

M. extorquens DM4 ProteomeDifferential proteomic analysis of cultures grown with DCM andwith methanol elucidates growth metabolism in the presence ofDCM.

[26]

M. extorquens CM4 Proteome andgenome

Comparison of growth with one-carbon substrates: chloromethaneand methanol, reporting the genes required for chloromethaneutilization.

[140]

M. extorquens AM1 Proteogenome Refined the annotation of protein coding genes and discover genesinM. extorquens AM1 genome.

[132]

M. extorquens AM1 MetabolomicAnalyze the metabolites produced during single carbon(methanol) and multicarbon (succinate) growth, providing clues tonew pathways that are specifically linked to C1 metabolism.

[136]

M. extorquens AM1 Metabolomic

Metabolites produced byM. extorquens AM1 grown on two carbonsources, ethylamine (C2) and succinate (C4) using LC-based andGC-based methods showing differences in in pathways linked toC2 and C4 metabolism.

[137]

M. extorquens AM1 TranscriptomeValidate a microarray plataform comparing genes expressedduring single carbon (methanol) and multicarbon (succinate)growth, pointing candidate genes in C1 metabolism.

[133]

M. extorquens AM1 Transcriptome Elucidates the regulation of general stress regulator (PhyR) usingmicroarray.

[139]

M. extorquens AM1 eDM4 Genome comparison

Genome comparison ofM. extorquens strain AM1 (referencestrain) and the dichloromethane-degrading strain DM4concluding that rearrangements and horizontal gene transferresulted in a great genomic plasticity.

[128]

Methylobacteriumstrains

Genomeannouncement

SixMethylobacterium strains adapted to different plant-associatedniches and environmental:M. extorquens (PA1, CM4. BJ001),M.radiotolerans (JCM2831),M. nodulans (ORS2060), andMethylobacterium sp. 4-46

[130]

M. mesophilicum SR1.6/6 Genomeannouncement

Endophytic bacterium isolated from a surface-sterilized Citrussinensis.

[127]

Methylobacterium sp.GXF4

Genomeannouncement

A xylem-associated bacterium isolated from Vitis vinifera L.grapevine.

[43]

influenced by plant genotype or by interactions with otherassociated microorganisms [6, 34].

Vuilleumier et al. [128] analyzed the genomes of twodifferentMethylobacterium strains (AM1 andDM4), showingthat there were variations in the numbers of insertionelements (IS) and in the organization of the genes, especiallythose associated with methanol (C

1) metabolism. Based on

these results, the authors suggested that IS is the mainmechanism for Methylobacterium evolution. On the otherhand, a recent study compared the M. extorquens strain PA1isolated from Arabidopsis plants to the well characterizedM. extorquens AM1 strain, showing that the CG contents

of PA1 and AM1 strain are quite similar, 68.2% and 68.5%,respectively. Moreover, 90 genes known to be involved withmethylotrophy presented more than 95% of identity betweenthese two strains at the amino acid level. Thus, the authorssuggested that these two strains have similar specificmodulesduring C1 growth; however, a different growth rate wasobserved when they used different substrates, which mayreflect an adaptation to the niche from which it was isolated[129]. There is a core of conserved genes in the genomes ofthe Methylobacterium genus, which was shown by a studythat sequenced six different Methylobacterium strains andobserved that five of the six strains showed conserved genes


involved in photosynthesis, including genes encoding thelight-harvesting complex and genes involved in the biosyn-thesis of bacteriochlorophyll and carotenoids [130], where thecore genome consists of 2010 genes and the pan genome of14,674 genes [40].

Sequenced genomes can be used to identify molecularmechanisms related to plant-Methylobacterium or microbe-Methylobacterium interactions. Kwak et al. [40] comparedthe genomes of nine Methylobacterium strains and reportedthat these strains could be divided into three groups withdistinguishable features: the first group contains genes fornitrogen fixation (M. nodulans and Methylobacterium sp. 4-46); the second group has many genes related to plant growth(such as ACCdeaminase and phytase) and no nitrogen-fixinggenes (M. oryzae and M. radiotolerans); and the third group(M. extorquens) lacked these previous genes.

The genetic and biochemical mechanisms involved inplant-bacteria interactions remain poorly explained, evenwith many sequenced genomes in databases; one of the moststudied bacteria of this genus, the AM1 strain, still needsto be explored, as reported by Ochsner et al. [131] in areview that gathers studies of AM1 strain. Kumar et al. [132]used a proteomics technique to propose a new annotationof a locus function in the AM1 strain. They predicted thatthe locus MexAM1 META1p1840, previously annotated ashypothetical, had a cytochrome C3 heme-binding domainand zinc finger domain of HSP40 (Table 2).

The differences between methanol and succinatemetabolisms were studied using different approaches. Usingmicroarray techniques, Okubo et al. [133] suggested aconnection between methylotrophy metabolism and ironand sulfur homeostasis. Using a proteomic approach, Boschet al. [134] and Laukel et al. [135] observed the differences inM. extorquensAM1 under methylotrophic growth conditionscompared to growth on succinate and observed that someproteins were induced depending on growth conditions. Themetabolite profiles under the previous conditions were alsoanalyzed by Guo and Lidstrom [136], who reported severaldifferent metabolites in cells grown on methanol and onsuccinate, although only 13 matched to the mass spectradatabase. Metabolomics studies of M. extorquens AM1 werealso performed in other conditions, comparing C2 andC4 metabolism, using ethylamine (C2) and succinate (C4)as carbon sources. Both liquid chromatography-tandemquadrupole mass spectrometry (LC-MS/MS) and two-dimensional gas chromatography-time-of-flight massspectrometry (GCxGC-TOF-MS) were used and were ableto validate the metabolites’ qualifications.The results showedthat the abundance of 20 intermediates varied under differentmetabolisms, evidence that there are differences not onlybetween the C1 and C4 but also between the C2 and C4pathways [137].

Schneider et al. [138] reported that the M. extorquensAM1 ethylmalonyl-CoA pathway functionally replaces theglyoxylate cycle (isocitrate lyase) during growth on acetate,suggesting that such an organism can adapt its metabolismto changes in carbon source availability. A proteomic studyof M. extorquens during the colonization of the phyllo-sphere of Arabidopsis thaliana described the upregulation

of proteins from the antioxidant system and underscoredthe increased expression of the PhyR regulator, which isimportant for the colonization of the phyllosphere [59].Later, Francez-Charlot et al. [139] elucidated PhyR regulationusing a transcriptomic analysis, showing that PhyR regulatesgene expression though protein-protein interactions and thatNepR negatively regulates PhyR by the sequestration of theECF sigma factor. Evidence of differences from other well-known general stress regulators, such as 𝜎𝑆 and 𝜎𝐵, suggeststhat Alphaproteobacteria has a novel mechanism of generalstress response.

In addition to their interactions with plants,Methylobac-terium strains are able to degrade several organic com-pounds, including dichloromethane and chloromethane [26,127, 140]. Different approaches comparing dichloromethane-degrading strains to the M. extorquens AM1 reference strainhave reported that rearrangements and horizontal gene trans-fer resulted in great genomic plasticity [128]. In addition,authors observed that the success of horizontal transferof the dcmA gene (which confers the ability to grow ondichloromethane) was not related to the phylogeny of theparental strain, but to the adaptation to the stress and themetabolic disruption resulting from the acquisition of a newenzyme or pathway [141].

In this way, Methylobacterium strains can be used toincrease plant growth (producing auxins and siderophores,fixing nitrogen, decreasing ethylene production, and solubi-lizing phosphorus), to inhibit plant pathogens and to inducesystemic resistance in plants. In addition, these strains can actin bioremediation processes that degrade toxic organic com-pounds, increasing plant tolerance and possibly increasingthe phytoremediation of inorganic compounds. All of thesecharacteristics show the bacterial potential in agriculture.

Several beneficial plant growth-promoting processes havealready been reported in Methylobacterium strains, as men-tioned above; however, more studies are needed to make itpossible to elucidate all of those interaction processes at thebiochemical and molecular levels. Genomic, transcriptomicand proteomic approaches enable us to study the structureand infer the functions of differentmetabolic pathways aswellas to understand some integrated aspects of microorganismbiology, that is, to correlate gene sequences, expressionpatterns, protein functions, and interactions. Thus, theseapproaches will provide us with essential clues to understandthe evolutionary history of biological systems and to supportbiotechnological applications in different areas of interest.

6. Concluding Remarks

There has been a lot of research in Methylobacterium genussince its first reported in 1976 [1], which was largely increasedafter the use of next generation sequencing technology,enabling us to study the genes present in the genome, theexpressed genes, and the proteins and metabolites producedin different conditions allowing us to understand somemechanisms involved during plant interaction, explaininghow these PPFM bacteria are able to induce plant growth,decreasing the establishment of plant pathogens and plantstress, as well as comprehend the role of these bacteria during


bioremediation of contaminated soils. Unfortunately, in thepresent review we were not able to include all publishedarticles of this subject; for more information, there areother Methylobacterium reviews [55, 131]. Moreover, thereare innumerousmicroorganism interactions occurring in theplant environment that still need to be elucidated, requiringmore research.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

This work was supported by a grant from the Founda-tion for Research Assistance, Sao Paulo State, Brazil (Proc.2010/07594-5 and 2012/24217-6). The authors thank FAPESPfor fellowships to Manuella Nobrega Dourado and AlineAparecida Camargo Neves and CNPq for the Daiene SouzaSantos Fellowship and the Welington Luiz Araujo ResearchFellowship.

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